Our goal is to discover the molecular mechanisms by which integrins sense and transduce mechanical cues. Integrins are heterodimeric transmembrane proteins that link the cell's cytoskeleton to the extracellular matrix (ECM). Cells use integrins to migrate, exert force on their surroundings, and to sense the physical properties of the ECM. This latter property, termed mechanotransduction, is particularly important in human health and disease. Physical tension transmitted through integrins activates intracellular signaling that in turn exerts profound effects on processes as diverse as immune function, stem cell differentiation, and cancer cell metastasis. Despite this great physiological and medical importance, the physical mechanisms by which integrins sense mechanical force are not known. We aim to close this fundamental gap in our understanding of cell biology. In published work, we have developed F?rster resonance energy transfer (FRET) based molecular tension sensors (MTSs) that report on the mechanical tensions experienced by individual integrins in living cells. We have since combined MTSs and superresolution light microscopy to, for the first time, map force transmission within integrin adhesions with nanometer spatial resolution. The qualitatively new capabilities of MTS-based imaging allow us to tackle two fundamental questions in integrin biology that until now could not be directly addressed. In Aim 1, we will determine the physical mechanisms by which integrins sense mechanical tension. In particular, we will examine the overarching hypothesis that different integrin classes sense tension via fundamentally different mechanisms, and that these differences allow the cell to sense mechanical stimuli over a wide range of forces and timescales. In Aim 2, we will characterize the force transducing and sensing machinery in micron-sized integrin assemblies, termed focal adhesions (FAs), for the first time. Specifically, we will test the hypothesis that FAs contain highly coordinated, force-sensing microdomains, a prediction that cannot be tested using conventional techniques. This work will transform our understanding of cellular mechanotransduction by uncovering the molecular assemblies and biophysical mechanisms by which cells sense and transduce mechanical signals. More broadly, the mechano-responsiveness and compositional complexity that characterize FAs are also present in many other cellular structures. The conceptual and technical approaches developed in this project have the capacity to transform multiple fields of research by introducing powerful new single-molecule biophysical measurements in the context of intact, living cells.